In recent years, enzymes have been used for the treatment of blood to alleviate the symptoms of several diseases. However, the release of enzymes into the blood stream not only inhibits control over the amount of enzyme activity, but in case of some enzymes can also be toxic. To solve these problems, enzymes have been immobolized onto an insoluble matrix over which an extracorporal blood stream is passed over the matrix to allow the immobilized enzymes to act, such as by cleaving L-asparagine into ammonia and aspartic acid. Enzyme immobilization also has been shown to increase enzyme stability.
As a more specific example, the enzyme L-asparaginase, which hydrolyzes asparagine to aspartic acid and ammonia, has demonstrated antineoplastic activity in acute lymphocytic leukemia in man. The mechanism of the soluble enzyme's activity has been related to the depletion of blood L-asparagine levels resulting in the death of leukemic cells lacking endogenous synthetic capacity , for that amino acid. L-asparaginase has also been shown to have immunsuppressive properties and is useful as a treatment with on organ transplants. Unfortunately, free L-asparaginase has a variety of toxic side effects related to hypersensitivity and the inhibition of protein synthesis which precludes administration of the soluble enzyme by injection. Therefore, it is desirable to immobolize such an enzyme to obtain its useful properties while diminishing or eliminating its undesirable effects.
Attempts have been made for immobilizing biochemically active enzymes on or within various types of matrices using various types of linking agents. However, these attempts have not been entirely successful. Among the problems encountered are a short shelf life for the matrix bound enzyme, leakage of enzyme from the matrix, a low enzyme holding capacity for of certain supports such as silica, incompatibility with blood flow because of such problems as platelet aggregation, and reduced enzyme activity because of excessive linkage to the enzyme or interference of substrate and enzyme product flow through the matrix.
Excessive activation of the matrix by the linking agent increases the probability of multiple site bonding to the enzyme which quite frequently results in enzyme inactivation. Certain prior methods of retaining enzymes on a matrix have also resulted in large increase in the apparent Michaelis constant ("K.sub.M "), indicating a substantial decrease in substrate affinity.
Accordingly, it would be desirable to provide a bio-artificial organ that avoids the deficiencies of the prior art and provides a biochemically active enzyme that remains active while being stably linked to a support matrix. The present invention provides such a bio-artificial organ.